Hedgehog Signaling Pathway PCR Array

NOTE: To access content of RT² Profiler PCR Arrays (Prior to May 25, 2012), please click here. Please NOTE: The RT² Profiler PCR Array System was upgraded to Version 4.0 on May 25, 2012. This system has replaced the Version 3.0 system with regards to content and assay design, with the Version 3.0 products set for discontinuation effective December 1, 2012.

The Mouse Hedgehog Signaling RT² Profiler™ PCR Array profiles the expression of 84 key genes involved in the hedgehog signaling pathway. The hedgehog family members, including Sonic hedgehog (Shh), are the most well-known morphogens involved in the developmental pattern formation of various organs, such as the nervous system, muscle, the heart and the lungs. Hedgehog signaling has also been implicated in the development of several cancers. The array includes hedgehog family members, hedgehog receptors, and other associated proteins. The array also includes key genes involved in cell differentiation and multi-cellular organism development. Using real-time PCR, you can easily and reliably analyze expression of a focused panel of genes related to hedgehog signaling with this array.

"If you have access to a real-time PCR instrument, you can use PCR Arrays"

RT² Profiler™ PCR Arrays are the most reliable and sensitive gene expression
profiling technology for analyzing a panel of genes in signal
transduction pathways, biological process or disease related gene networks. The PCR Arrays can be
used for research on cancer, immunology, stem cells,
toxicology, biomarker discovery and validation, and phenotypic analysis of
cells and transgenic animals. See the complete list of PCR Arrays.

The PCR array is a set of optimized real-time PCR primer assays on 96-well or
384-well plates for pathway or disease focused genes as well as appropriate RNA
quality controls. The PCR array performs gene expression analysis with real-time PCR
sensitivity and the multi-gene profiling capability of a microarray. Simply mix
your cDNA template with the appropriate ready-to-use PCR master mix, aliquot
equal volumes to each well of the same plate, and then run the real-time PCR
cycling program. (Download user manual)

Layout and Controls: The PCR Arrays are available in both 96- and
384-well plates and are used to monitor the expression of 84 genes related to a
disease state or pathway plus five housekeeping genes. Controls
are also included on each array for genomic DNA contamination, RNA quality, and
general PCR performance

You can easily perform a PCR Array experiment in your own laboratory, or send
your samples to us and take advantage of our PCR
Array Services.

Performance Data Sensitivity:
The complete PCR Array System yields a greater-than 85 percent present call with
as little as 25 ng as much as 5 µg of total RNA from a pathway representing
genes expressed at a lower level (inflammatory cytokines and receptors).

Figure 2:

PCR Arrays Let You See More Genes
with Less RNA
Different amounts of universal total RNA were characterized using the
Human Inflammatory Cytokines and Receptors PCR Array, and the percentage
of detectable genes was calculated for each RNA amount.
As little as 25 ng total RNA yields greater than an 80% positive call,
even for cytokines expressed at very low levels.

Specificity
The complete PCR Array System, with high quality input RNA, is guaranteed to
yield single bands of the predicted size without primer dimers or other
secondary products thus providing the most accurate real-time PCR results
possible.

Figure 4:

PCR Arrays Amplify A Single Gene-Specific Product in Every Reaction.
Universal total RNA was characterized on the TGFβ / BMP Signaling Pathway PCR Array, followed by dissociation (melt) curve analysis.
PCR Arrays specifically detect individual genes despite the expression of related gene family members in the same RNA sample.

Application Data

Cancer Research:
To ascertain the oncogenic route that two different human breast tumors have
taken, the relative expression level of cancer- and adhesion-related genes in
normal and two different cancerous tissues were compared.

ECM & Adhesion PCR Arrays Revealed Up- and Down-Regulated Genes in Breast Cancer
Total RNA from normal human breast and a human breast tumor were characterized in technical triplicates, and the relative expression levels for each gene in the two samples are plotted against each other in the Scatter Plot.
Genes encoding the matrix metallopeptidases (MMP3 & MMP9) and their inhibitors (TIMP3) are up-regulated, while genes encoding integrins (ITGB3 & ITGB4) are down-regulated, by at least three-fold (outside the silver field) in breast tumors relative to normal tissue.

Toxicology Research:Rezulin (Troglitazone or "Tro" or "T"), a glitazone
PPAR-gamma agonist, was approved for treatment of type 2 diabetes mellitus, but was
withdrawn from the market due to idiosyncratic liver toxicity. Two similar
drugs, Avandia (Rosiglitazone or "Rosi" or "R") and Actos (Pioglitazone
or "Pio" or "P"), are considered to be safe treatments for
the same condition. The expression profile of key drug metabolism genes should
be different in cells treated with Rezulin versus those treated with Avandia and
Actos.

Hepatocellular carcinoma HepG2 cells were treated at 80% cell confluence with
these three drugs (100 µM, Cayman Chemical) or a DMSO vehicle control for 24 h.
RNA isolated using the ArrayGrade™ Total RNA Isolation Kit was used to
characterize gene expression with the Human Drug Metabolism and
Stress & Toxicity PathwayFinder™ RT² Profiler™ PCR Arrays and
RT² SYBR Green / Fluorescein PCR master mix on the Bio-Rad iCycler®.

Figure 6:

Stress & Toxicity PathwayFinder™ PCR Array Uncovered Idiosyncratic Mechanisms of Action for Liver Toxicity Caused by 3 PPARγ Agonists.
RNA from HepG2 cells treated with three different glitazone PPARγ agonists for type 2 diabetes mellitus was characterized, and the results were compared to that of a vehicle (DMSO) control.
A withdrawn drug with idiosyncratic liver toxicity (Rezulin) induces very different changes in the expression of stress-related genes than two safer drugs still on the market (Avandia and Actos).